EP0849360A1 - Gène de la glycosyl-transferase mono fonctionnelle de Staphylococcus aureus - Google Patents

Gène de la glycosyl-transferase mono fonctionnelle de Staphylococcus aureus Download PDF

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EP0849360A1
EP0849360A1 EP97310207A EP97310207A EP0849360A1 EP 0849360 A1 EP0849360 A1 EP 0849360A1 EP 97310207 A EP97310207 A EP 97310207A EP 97310207 A EP97310207 A EP 97310207A EP 0849360 A1 EP0849360 A1 EP 0849360A1
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Prior art keywords
mtg
seq
nucleic acid
host cell
vector
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German (de)
English (en)
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Jo Ann Hoskins
Stanley Richard Jaskunas, Jr.
Robert Brown Peery
Paul Luther Skatrud
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/82—Proteins from microorganisms
    • Y10S530/825—Bacteria

Definitions

  • This invention relates to recombinant DNA technology.
  • the invention pertains to the cloning of a gene, mtg, encoding a novel monofunctional glycosyltransferase, from Staphylococcus aureus and the use of said gene and its encoded protein in a screen for new inhibitors of bacterial cell wall biosynthesis.
  • the bacterial cell wall comprises a peptidoglycan layer which provides mechanical rigidity for the bacterium.
  • the peptidoglycan layer is composed of a sugar backbone (alternating residues of N-acetylglucosamine and N-acetylmuramic acid are polymerized through a transglycosylation reaction) attached to a pentapeptide (also referred to as "stem peptide") containing D and L amino acid residues. Adjacent stem peptide residues are covalently crosslinked during maturation of the peptidoglycan.
  • a lipid-linked disaccharide-pentapeptide is translocated across the cytoplasmic membrane, exposing the pentapeptide sidechains to the cell surface.
  • the fully mature peptidoglycan structure is obtained following transglycosylation and transpeptidation enzymatic reactions.
  • Several enzymes appear to be involved in the transglycosylation and transpeptidation polymerizaion reactions, most notably the bifunctional high molecular weight PBPs.
  • transglycosylation activity is also found in monofunctional enzymes known as monofunctional glycosyltransferases (MTG's).
  • MTG monofunctional glycosyltransferases
  • the present invention provides, inter alia, isolated nucleic acid molecules that encode an MTG from Staphylococcus aureus.
  • the invention also provides protein products encoded by the gene, in substantially purified form.
  • Having the cloned mtg gene of Staphylococcus aureus enables the production of recombinant MTG protein and derivatives thereof for the implementation of assays and screens to identify new inhibitory compounds targeted at the peptidoglycan biosynthetic pathway.
  • the present invention relates to isolated gene mtg that encodes novel Staphylococcus aureus MTG, said gene comprising the nucleotide sequence identified as SEQ ID NO. 1.
  • the present invention relates to a novel protein molecule, MTG, wherein said protein molecule comprises the sequence identified as SEQ ID NO. 2.
  • the present invention relates to a soluble form of MTG (designated MTG S ) wherein MTG S comprises amino acid residues 68 through 269 of SEQ ID NO.2.
  • the present invention relates to a ribonucleic acid molecule encoding MTG protein, said ribonucleic acid molecule comprising the sequence identified as SEQ ID NO. 3 or fragment thereof.
  • the present invention relates to a recombinant DNA vector that incorporates the Staphylococcus aureus mtg gene in operable linkage to gene expression sequences enabling said mtg gene to be transcribed and translated in a host cell.
  • the present invention relates to homologous or heterologous host cells that have been transformed or transfected with a vector carrying the cloned mtg gene from Staphylococcus aureus such that said gene is expressed in the host cell.
  • the present invention relates to a method for identifying compounds that bind and/or inhibit the enzymatic activity of the Staphylococcus aureus MTG protein or fragment thereof.
  • Plasmid pPSR-23 useful for high level expression of the Staphylococcus aureus mtg S gene of the present invention in the heterologous procaryotic host cell Eschericia coli.
  • mtg refers to the Staphylococcus aureus genomic DNA sequence encoding MTG and fragments thereof.
  • mtg S refers to a portion of mtg that encodes MTG S comprising nucleotide residues 202 through 807 of SEQ ID NO.1.
  • MMG as used herein may refer to the native monofunctional glycosyltransferase or to a portion thereof.
  • cleavage or “restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (viz. sequence-specific endonucleases).
  • restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements are used in the manner well known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can readily be found in the literature.
  • fusion protein denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain.
  • “Functional domain” refers to a region of a protein having one or more distinct biological functions, for example, enzymatic activity, transmembrane anchoring, DNA binding, etc.
  • a functional domain comprises a sequence of amino acids, the length of which and the identity of amino acid residues therein, may or may not be critical to said function.
  • Plasmid refers to an extrachromosomal genetic element.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • Recombinant DNA cloning vector refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • recombinant DNA expression vector refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present to enable transcription of the inserted DNA.
  • vector refers to a nucleic acid compound used for introducing exogenous DNA into host cells.
  • a vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.
  • complementary refers to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding in double stranded nucleic acid molecules.
  • the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
  • isolated nucleic acid compound refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.
  • a “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.
  • promoter refers to a DNA sequence which directs transcription of DNA to RNA.
  • a “probe” as used herein is a labeled single-stranded nucleic acid compound of greater than 5 nucleotide residues that has the potential to hybridize with another nucleic acid compound.
  • hybridization refers to the phenomenon by which a single-stranded nucleic acid molecule joins with a complementary single-stranded nucleic acid molecule through nucleotide base pairing.
  • Selective hybridization refers to hybridization that occurs under conditions of high stringency. The extent of hybridization depends upon a number of variable and constant parameters, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.
  • stringency refers to hybridization conditions. High stringency conditions disfavor non-homologous basepairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by changing the temperature and salt concentration.
  • Transglycosylation refers to an enzymatic reaction in which the sugar residues of lipid-linked disaccharide pentapeptide molecules are polymerized during the formation of the peptidoglycan structure of the bacterial cell wall.
  • the mtg gene (SEQ ID NO.1) of the present invention encodes a novel MTG enzyme of Staphylococcus aureus (SEQ ID NO. 2).
  • the mtg gene disclosed herein comprises a DNA sequence of 807 nucleotide base pairs (SEQ ID NO. 1). There are no intervening sequences.
  • numerous "silent" substitutions of nucleotide base pairs could be introduced into the sequence identified as SEQ ID NO. 1 without altering the identity of the encoded amino acid(s) or protein product. All such substitutions are intended to be within the scope of the invention.
  • the MTG protein defined by SEQ ID NO.2 comprises a membrane-bound protein of 269 amino acid residues.
  • the MTG protein of the present invention may be modified by deletion of amino acid residues 1 through 67 at the amino terminal end. Deletion of this region results in removal of the trans-membrane region and the production of a soluble form, MTG S which retains the transglycosylase domain of the native enzyme.
  • Another modified form of MTG, which retains the transmembrane region comprises amino acid residues 17 through 269 of SEQ ID NO.2.
  • the gene of the present invention may be obtained by a plurality of applicable genetic and recombinant DNA techniques including, for example, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis.
  • PCR polymerase chain reaction
  • de novo DNA synthesis See e.g. , J.Sambrook et al. Molecular Cloning , 2d Ed. Chap. 14 (1989)).
  • the mtg gene of Staphylococcus aureus comprising the present invention or fragment thereof could be isolated by PCR amplification of Staphylococcus aureus genomic DNA or cDNA using oligonucleotide primers targeted to any suitable region of SEQ ID NO. 1.
  • Methods for PCR amplification are widely known in the art. See e.g. PCR Protocols: A Guide to Method and Application , Ed. M. Innis et al., Academic Press (1990).
  • the amplification reaction comprises genomic DNA, suitable enzymes, for example Taq DNA polymerase, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT). A positive result is determined by detecting an appropriately-sized DNA fragment following agarose gel electrophoresis.
  • One embodiment of the present invention relates to the substantially purified MTG protein or fragment thereof, for example MTG S .
  • amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Patent 4,617,149, incorporated herein by reference.
  • polypeptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA) and synthesis cycles supplied by Applied Biosystems.
  • Applied Biosystems 430A peptide synthesizer Applied Biosystems, Foster City, CA
  • Protected amino acids such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.
  • the proteins of the present invention can also be produced by recombinant DNA methods using the cloned mtg gene or fragment thereof, as disclosed herein. Recombinant methods are preferred if a high yield of protein is desired. Expression of said cloned gene can be carried out in a variety of suitable host cells well known to those skilled in the art. In a recombinant method the mtg or mtg S gene or variants thereof are introduced into a host cell by any suitable means, well known to those skilled in the art.
  • chromosomal integration of a cloned mtg gene or variant thereof is within the scope of the present invention, it is preferred that the gene be cloned into a suitable extra-chromosomally maintained expression vector so that the coding region of the gene is operably linked to a constitutive or inducible promoter.
  • procaryotes are used for cloning DNA sequences and for constructing the vectors of the present invention.
  • Procaryotes may also be employed in the production of the MTG proteins of the present invention.
  • the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the procaryotic expression of foreign proteins.
  • Other strains of E. coli, bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, various Pseudomonas species and other bacteria, such as Streptomyces, may also be employed as host cells in the cloning and expression of the recombinant proteins of this invention.
  • Promoter sequences suitable for driving the expression of genes in procaryotes include b -lactamase [e.g. vector pGX2907, ATCC 39344, contains a replicon and b -lactamase gene], lactose systems [Chang et al., Nature (London), 275:615 (1978); Goeddel et al., Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATH1 (ATCC 37695) which is designed to facilitate expression of an open reading frame as a trpE fusion protein under the control of the trp promoter].
  • vector pGX2907 e.g. vector pGX2907, ATCC 39344, contains a replicon and b -lactamase gene
  • lactose systems Chang et al., Nature (London), 275:615 (1978
  • Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Still other promoters, such as that from bacteriophage T7, whose nucleotide sequences are generally known, enable one of skill in the art to ligate such promoter sequences to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Still other promoters are useful for gene expression in S.
  • Promoters for use in bacterial systems will also contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired polypeptides. These examples are illustrative rather than limiting.
  • yeast Saccharomyces cerevisiae is the most commonly used eucaryotic microorganism.
  • a number of other yeasts such as Kluyveromyces lactis are also suitable.
  • the plasmid YRp7 (ATCC-40053), for example, may be used. See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman et al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980).
  • Plasmid YRp7 contains the TRP1 gene which provides a selectable marker for use in a trpl auxotrophic mutant.
  • An expression vector carrying the cloned mtg or mtg S of Staphylococcus aureus or fragment thereof is transformed or transfected into a suitable host cell using standard methods.
  • Cells that contain the vector are propagated under conditions suitable for expression of the encoded MTG.
  • suitable growth conditions would incorporate an appropriate inducer.
  • Recombinantly-produced MTG S or MTG protein may be purified from cellular extracts of transformed cells by any suitable means.
  • Recombinantly-produced MTG that contains the N-terminal portion of the protein is expected to be localized in the host cell membrane.
  • recombinant MTGs may also be recoverable from cell extracts and cell membranes by any suitable means, well known to those skilled in the art.
  • the gene encoding the MTG or MTG S of the present invention is modified at the 5' end to incorporate several histidine residues at the amino terminal end of the respective protein molecules.
  • the "histidine tag” method enables a simplified protein purification known as "immobilized metal ion affinity chromatography” (IMAC), essentially as described in U.S. Patent 4,569,794, which hereby is incorporated by reference.
  • IMAC immobilized metal ion affinity chromatography
  • inventions of the present invention comprise isolated nucleic acid sequences.
  • proteins of the invention can be encoded by a multitude of different nucleic acid sequences. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.
  • Nucleic acid sequences that encode SEQ ID NO:2 or subregion therein may be produced using synthetic methods.
  • the synthesis of nucleic acids is well known in the art. See, e.g., E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana, Methods in Enzymology , 68:109-151 (1979).
  • the DNA segments corresponding to the mtg or related gene sequence mtg S could be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) which employ phosphoramidite chemistry.
  • phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention.
  • phosphotriester chemistry See, e.g., M.J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach , (1984).]
  • the DNA sequence comprising a portion or all of SEQ ID NO:1 can be generated from Staphylococcus aureus genomic DNA using suitable oligonucleotide primers complementary to SEQ ID NO:1 or region therein, essentially as described in U.S. Patent No. 4,889,818, which hereby is incorporated by reference.
  • suitable protocols for performing the PCR are widely known and are disclosed in, for example, PCR Protocols: A Guide to Method and Applications , Ed. Michael A. Innis et al., Academic Press, Inc. (1990).
  • ribonucleic acids of the present invention may be prepared using the polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically using RNA polymerase to transcribe a suitable DNA template.
  • RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5' end of the template to be transcribed. See, J. Sambrook, et al., supra, at 18.82-18.84.
  • This invention also provides nucleic acids, RNA or DNA, that are complementary to SEQ ID NO:1 or SEQ ID NO:3.
  • the present invention also provides probes and primers useful for a variety of molecular biology techniques including, for example, hybridization screens of genomic or subgenomic libraries.
  • a nucleic acid compound comprising SEQ ID NO:1, SEQ ID NO:3 or a complementary sequence thereof, or a fragment thereof, and which is at least 18 base pairs in length, and which will selectively hybridize to Staphylococcus aureus DNA or mRNA encoding the MTG or fragment thereof of the present invention, is provided.
  • the 18 or more nucleotide bases are DNA.
  • These probes and primers can be prepared by enzymatic methods well known to those skilled in the art ( See e.g. Sambrook et al. supra ). In a most preferred embodiment these probes and primers are synthesized using chemical means as described above.
  • Another aspect of the present invention relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. Many of the vectors encompassed within this invention are described above.
  • the preferred nucleic acid vectors are those that comprise DNA.
  • the most preferred recombinant DNA vectors comprise the isolated DNA sequence, SEQ ID NO:1.
  • Plasmid pPSR-23 is an especially preferred DNA vector for expressing the soluble form of the MTG of this invention in E. coli.
  • cloning vector or expression vector depends upon a number of factors including the availability of restriction enzyme sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into the host chromosome), the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another), and the number of copies of the gene to be present in the host cell.
  • readily assayable or selectable markers e.g., antibiotic resistance and metabolic markers of one type and another
  • Vectors suitable to carry the nucleic acids of the present invention comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like.
  • the most preferred vectors are plasmids.
  • inducible promoters are preferred because they enable high level, regulatable expression of an operably linked gene.
  • inducible promoters that respond to a variety of inducers, for example, carbon source, metal ions, heat, and others.
  • the amount of nucleic acid or protein to be produced dictates, in part, the selection of the expression system.
  • the addition of certain nucleotide sequences is useful for directing the localization of a recombinant protein. For example, a sequence encoding a signal peptide preceding the coding region of a gene, is useful for directing the extra-cellular export of a resulting polypeptide.
  • Host cells harboring the nucleic acids disclosed herein are also provided by the present invention.
  • a preferred host is E. coli that has been transfected or transformed with a vector that comprises a nucleic acid of the present invention.
  • the present invention also provides a method for constructing a recombinant host cell capable of expressing SEQ ID NO:2, or the soluble form thereof, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector that comprises an isolated DNA sequence which encodes SEQ ID NO:2 or fragment thereof.
  • the preferred host cell is any strain of E. coli that can accomodate high level expression of an exogenously introduced gene.
  • Preferred vectors for expression are those which comprise SEQ ID NO:1.
  • An especially preferred expression vector for use in E. coli is pPSR-23, which comprises nucleotide residues 202 through 807 of SEQ ID NO:1. (See Figure).
  • Transformed host cells may be cultured under conditions well known to skilled artisans such that a recombinant protein is expressed, thereby producing in the recombinant host cell the MTG or MTG S of the instant invention.
  • the instant invention provides a screen for identifying compounds that inhibit the enzymatic activity of MTG or MTG S or fragment thereof, said screen comprising the steps of:
  • the MTG or MTG S used in these experiments is preferably substantially purified as described herein.
  • An alternative method for purifying membrane-bound MTG would comprise extraction from solubilized membrane preparations of cells transformed with the cloned mtg gene. Solubilized membranes are prepared according to well known methods.
  • the substrate for an MTG transglycosylase assay can be made according to art-recognized methods ( See e.g. DiBerardino et al. FEBS Letters, 392, 184-88 (1996).
  • the lipid precursor substrate can be prepared from Staphylococcus aureus membranes, or from the membranes of any other suitable bacteria, UDP-Mur-Nac-pentapeptide, and UDP-N-acetyl- [ 14 C]glucosamine (Amersham, Buckinghamshire, UK).
  • Transglycosylase activity is measured by the production of the peptidoglycan polymerization product essentially by mixing the substrate with a source of MTG and monitoring the amount of [ 14 C]-label in the peptidoglycan.
  • the screening system described above provides a means to determine compounds that interact with the MTG of the present invention and which may interfere with peptidoglycan biosynthesis.
  • This screening method may be adapted to automated procedures such as a PANDEX® (Baxter-Dade Diagnostics) system, allowing for efficient high-volume screening for potential inhibitory agents.
  • Plasmid pPSR-23 (See Figure) is an approximately 6300 base pair expression vector suitable for expressing a modified mtg S in procaryotic host E. coli.
  • This plasmid contains an origin of replication (Ori), an ampicillin resistance gene (Amp), a T7 promoter, and a chelating peptide and Factor Xa site in operable linkage to the coding region of said mtg S gene.
  • the chelating peptide and Factor Xa site are engineered onto the amino terminal end of the recombinant MTG S in order to simplify protein purification by providing a "his tag.” (See Example 4).
  • the parent plasmid of pPSR-23, pET16B (obtained from Novogen, Madison, WI), was digested with endonucleases Nde I and BamHI . Digested pET16B was ligated to a DNA fragment bearing Nde I and BamHI sticky ends, comprising a modified mtg S gene.
  • the mtg S gene ligated into pPSR-23 encodes amino acid residues 68 through 269 of SEQ ID NO.2.
  • the mtg S gene carried on pPSR-23 is most conveniently produced by PCR using standard methods and oligonucleotide primers targeted to the 5' and 3' ends of SEQ ID NO.1.
  • the primer for synthesis at the 5' end of the gene is constructed to contain an NdeI site while the primer for synthesis at the 3' end of the gene is constructed to contain a BamH1 site for cloning into pET16B.
  • Example 1 The plasmid construction method outlined in Example 1 is followed to construct a vector for expressing MTG comprising amino acid residues 17 through 269 of SEQ ID NO.2 in a heterologous host such as E. coli.
  • Synthesis of the mtg gene used herein is most conveniently carried out by PCR on genomic DNA from S. aureus. Synthesis is primed at the 5' end of the gene starting at nucleotide position 49 of SEQ ID NO.1 using an appropriately synthesized oligonucleotide primer. This site comprises a natural NdeI site useful for cloning into plasmid pET16B.
  • Synthesis at the 3' end of the mtg gene is primed using a primer constructed to contain a BamH1 cloning site and targeted to nucleotide residues extending through nucleotide position 807 at the 3' end of SEQ ID NO.1.
  • Expression plasmid pPSR-23 was transformed into E. coli BL21 (DE3)pLys5(F - ompT[lon]hsd S r B - m B - ) using standard methods ( See e.g. Sambrook et al. Supra ). Transformants chosen at random were tested for the presence of pPSR-23 by agarose gel electrophoresis using quick plasmid preparations. Id. Transformants were grown overnight at 37_ C in LB medium supplemented with 100 mg/ml ampicillin. The overnight culture was diluted into fresh LB medium and allowed to grow to an O.D. 600 of 0.6 to 0.8. At that point, expression of the vector-bound mtgS gene was induced by adding 1.0 mM IPTG for a period of 4 hours. The induced-culture was then pelleted by centrifugation in preparation for protein purification (See Example 4).
  • the recombinant cell pellet isolated as described in the last step of Example 3, was resuspended in 60 ml of 20 mM potassium phosphate, pH 7.5.
  • the cells were disrupted by passage through a French press, producing a cell extract that was centrifuged at 150,000 x g for 1 hour.
  • the MTG S protein contained in the extract was purified by immobilized metal ion affinity chromatography (IMAC), essentially as described in US Patent 4,569,794, the entire contents of which is hereby incorporated by reference. Briefly, the IMAC procedure involved adding to the protein sample the following components at the indicated final concentrations: 0.5M NaCl, 5 mM imidazole.
  • the sample was loaded onto a Chelating Sepharose Fast Flow column (Pharmacia, 10 ml bed volume) and the column washed twice with 35 ml each of 20 mM Tris, pH 8, 0.5 M NaCl and 5 mM imidazole; 20 mM Tris, pH 8, 0.5 M NaCl and 60 mM imidazole.
  • the bound protein was eluted from the column with 20 mM Tris, pH 8, 0.5 M NaCl, 1 M imidazole.
  • Radiolabelled lipid precursor for use as substrate is prepared as described in H. Hara and H. Suzuki FEBS Lett. 168, 155-60 (1984). Peptidoglycan synthesis activities are determined in 50 ml reactions containing 50 mM PIPES, pH 6.1, 10 mM MgCl 2 , 0.2 mM DTT, 1 mM ATP, 26% DMSO, MTG or MTG S sample and 14 C-labelled lipid precursor. The reaction is incubated for 30 minutes at room temperature and filtered through hydrophilic Durapore PVDF membranes (0.65 mm; Millipore, Bedford, MA). Under these conditions the synthesized peptidoglycan is retained while the unincorporated labeled substrate is washed through using 0.4 M ammonium acetate in methanol. The filter-bound radioactivity is determined by scintillation counting.
  • Inhibition studies are carried out using the same reaction conditions described except that compounds to be studied for inhibitory activity are added to a final concentration between 1 mM and 10 mM.

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EP97310207A 1996-12-20 1997-12-16 Gène de la glycosyl-transferase mono fonctionnelle de Staphylococcus aureus Withdrawn EP0849360A1 (fr)

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US6143868A (en) * 1996-12-20 2000-11-07 Eli Lilly And Company Monofunctional glycosyltransferase of Staphylococcus aureus

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WO2003016908A2 (fr) * 2001-08-17 2003-02-27 The Trustees Of Princeton University Gene et produits genetiques conferant une susceptibilite aux antibiotiques derives de la vancomycine, methodes et titrages d'identification d'antibiotiques de glycopeptides bifonctionnels les utilisant
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US5922540A (en) 1999-07-13
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US6143868A (en) 2000-11-07
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